HPK1 is a mammalian Ste20-related serine/threonine kinase, which has been shown to regulate NF?B and JNK pathways in hematopoietic cells. Our lab is the first to discover that HPK1 is a novel tumor suppressor in pancreatic ductal carcinoma (PDC). Our data show that HPK1 is expressed in normal pancreatic ductal cells, but is lost in >95% PDCs. Loss of HPK1 occurs in early PanINs and is strongly associated with the progression from early PanINs to PDC both in human and Kras PDC models. Restoring HPK1 expression in PDC cells causes cell cycle arrest and growth inhibition, which is due in part to the stabilization of p21 and p27 (3). In addition, we have also demonstrated that loss of HPK1 in PDC is mediated by CUL7/Fbxw8 ubiquitin ligase through 26S proteasome, which requires HPK1 kinase activity and autophosphorylation. Targeted degradation of HPK1 by CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain HPK1 activity (4). To further examine the mechanisms of HPK1 regulation, we showed that COP9 signalosome 6 (CSN6) regulates HPK1 through CUL7/Fbxw8 ubiquitin ligase. Furthermore, our preliminary data showed that HPK1 down- regulates Axl via endocytosis and functions as a negative regulator of Ras through p120GAP and that loss of HPK1 plays a critical role in the development of PDC. Given the fact that both Kras mutations and loss of HPK1 occurs in early PanINs, it would be extremely important to examine the mechanisms of HPK1 degradation and its downstream signaling to identify key molecular targets involved in the progression of PanINs. The primary objectives of this proposal are to examine the mechanisms by which CSN6 regulates the functions of CUL7/Fbxw8 ubiquitin ligase and HPK1 and to examine the mechanisms and tumor suppressor functions of HPK1 in Axl, Ras signaling and Kras-driven development and progression of PanINs to PDC using mouse models. Our proposed studies will not only provide new insights into the regulation of CUL7/Fbxw8 ubiquitin ligase and HPK1 by CSN6 and the novel mechanisms by which HPK1 regulates p120GAP, Axl, and Ras signaling, but also establish HPK1 as a novel negative regulator of Ras signaling and a tumor suppressor in PDC. Through the dissection of novel pathways involved in HPK1 regulation combined with our new mouse models, we will identify molecular markers for early detection and develop novel mechanism-based strategy for PDC treatment by targeting CSN6-CUL7/Fbxw8-HPK1-Ras pathways. Therefore the proposed research is highly relevant to improving the outcome of PDC patients. We have three specific Aims: Specific Aim 1: To study the mechanisms by which HPK1 inhibits Ras signaling in pancreatic cancer Specific Aim 2: To examine the molecular mechanisms by which CSN6 regulates CUL7/Fbxw8 ubiquitin ligase and HPK1 in pancreatic cancer Specific Aim 3: To study the tumor suppressor function of HPK1 in antagonizing Kras signaling using KrasG12D pancreatic cancer mouse models